1. Field of the Invention
The present invention relates to a monoclonal antibody specifically recognizing a human liver-carboxylesterase 1 which can be used to objectively analyze an expression level of the human liver-carboxylesterase 1 from human clinical specimens, such as a tissue and the blood, a hybridoma cell line producing the antibody, and its use thereof.
2. Discussion of Related Art
A human carboxylesterase (hCE) may be mainly classified as isoform having different properties and structures such as a human carboxylesterase 1 (hCE1), a human carboxylesterase 2 (hCE2), and a human carboxylesterase 3 (hCE3). (Imai T, Drug Metab Pharmacokinet., vol. 21, pp. 173-185, 2006). The hCE1 is an enzyme generally biosynthesized in the liver and recognizing an acyl group of a hydrophilic molecule to decompose a chemical material absorbed into a tissue. However, the hCE2 is an enzyme generally expressed in the intestine, which is a separate protein having homology to the hCE1 of 47%, and reacting with an acyl group of a hydrophobic molecule absorbed into the intestine. The hCE3 is an enzyme generally expressed in the brain, which may also be a separate protein having homology to the hCE1 of 50%.
A liver-carboxylesterase 1 is an enzyme expressed in the liver, intestine, kidney, lung, heart, or macrophage, but is expressed in the liver 10 to 100 times higher than in the others. The liver-carboxylesterase 1 has three major roles: First, it has a function of xenobiotic metabolism which converts an inactive drug input into a human body into an active drug by modifying ester or amide structure of the inactive drug. For example, lovastatin is converted into an active form to reduce cholesterol, and serves to convert cocaine and heroin, which are toxic in the body, into non-toxic cocaethylene and morphine, respectively. Second, it can regulate cholesterol metabolism by decomposing and binding to an ester structure of cholesterol and a fatty acid in the body as needed, and third, it serves to regulate a function by directly binding to a protein, that is, C-reactive protein (CPR), recognizing the immunization of a macrophage in an endoplasmic reticulum (Redinbo M R., Biochem. Soc. Trans., 31, pp. 620-624, 2003; Redinbo M R., Drug Discov. Today., 10, pp. 313-325, 2005). Another property of the liver-carboxylesterase 1 is that its enzymatic activity is drastically decreased in a liver microsome of a rat in which a liver cancer is induced by a chemical material (Maki T., Jpn. J. Cancer Res., 82, pp. 800-806, 1991), and a mechanism thereof is not discovered yet.
Until now, a degree of activity of a liver-carboxylesterase 1 in vivo was analyzed as an enzyme titration value using a non-specific substrate of an esterase enzyme, such as p-nitrophenylphosphate or p-nitrophenylacetate. According to such a method, it is impossible to measure specific activity of only the liver-carboxylesterase 1, because albumin, acetylcholinesterase, butyrylcholinesterase, etc. having a function of an esterase in the blood also have the same activities (Li B., Niochem. Pharmacol., 70, pp. 1673-1684, 2005). Thus, a method capable of selectively and effectively analyzing a liver-carboxylesterase 1 protein from a large amount of specimens for a short time is needed, and such a method is enzyme immunoassay. The enzyme immunoassay is a method of inducing antigen-antibody binding through a reaction of an antibody binding to a desired protein (antigen), and quantifying a desired protein in a clinical specimen from coloring or fluorescence produced by a binding degree in a reaction to form a complex between an enzyme binding to an antibody and a substrate.
Recently, the inventors proved the presence of a liver-carboxylesterase 1 in a human plasma, and that an expression level of the liver-carboxylesterase 1 was increased by 2.8 times or more on average in the plasma of a liver cancer patient, compared to in the plasma of a normal person, while an expression level of the liver-carboxylesterase 1 was decreased in a liver cancer tissue, compared to that in a normal liver tissue (Na K. et al., Proteomics. 9, pp. 3989-3999, 2009). However, an antibody of the liver-carboxylesterase 1 used in the experiment is a commercially available polyclonal antibody (Abcam, ab1875), to which non-specific proteins can bind, and which is decreased in immunoprecipitation efficiency.